The anti-gastric ulcer effect of Gynostemma pentaphyllum Makino.
Gynostemma pentaphyllum is an oriental medicinal herb reputed to have broad-spectrum activities. The plant's principal saponin components are structurally similar to those found in ginseng plants and this similarity is assumed to be responsible for the claimed activities. The present study was undertaken to evaluate a G. pentaphyllum butanol fraction (GPB) for its anti-gastric ulcer activity using experimental models. Oral administration of the GPB at 200 and 400 mg/kg body wt. significantly inhibited gastric ulcer formation induced by indomethacin, HCl/EtOH and water-immersion restraint stress in rats. In pylorus-ligated rats, pretreatment with the GPB had no effect on gastric volume, pH or acidity output, thus indicating a lack of anti-secretory effect. In ethanol-induced ulcerated rats, gastric wall mucus and hexosamine content were markedly preserved by GPB pretreatment. The findings indicate that the butanol fraction of G. pentaphyllum possesses gastroprotective potential related to the preservation of gastric mucus synthesis and secretion.
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Keywords: Gynostemma pentaphyllum; Saponin; Gastric ulcers
Gynostemma pentaphyllum Makino (family Cucurbitaceae) is a perennial creeping herb growing wild in the mountainous regions of Japan, China and many other Aisan countries. It has been used in Chinese and Japanese folk medicine to alleviate various diseases and symptoms including hypertension, cough, migraine, insomnia and diabetes mellitus (Takemoto et al., 1983). In Thailand G. pentaphyllum has been found growing wild in the northern region (Hiranramdej and Maxwell, 1992). The plant has also been cultivated by private firms and the King's Royal Project for commercial purposes. Phytochemical studies of this plant have identified saponin glycosides (called gypenosides), which are structurally similar to those found in ginseng (Cui et al., 1999). Gypenosides III, IV, VIII, XII and malonyl gypenosides III and VIII are identical to ginsenosides [Rb.sub.1], [Rb.sub.3], Rd, [F.sub.2], and malonyl ginsenosides [Rb.sub.1] and Rd (Takemoto et al., 1983; Kuwahara et al., 1989) found in Panax ginseng C.A. Meyer (Korean ginseng). Furthermore, gypenoside VXVII was also isolated from P. quinquefolium, P. noto ginseng and P. japonicus (Morita et al., 1985). Due to this structural similarity, it is claimed that drinking a tea made of G. pentaphyllum leaves regularly could promote good health and lessen the severity of many disorders, including ulcers of the stomach. The objective of this work is thus to examine the anti-gastric ulcer activity of a G. pentaphyllum butanol (GPB) fraction, which includes saponins, in experimental animals and provide scientific data to substantiate the claims made.
Materials and methods
Preparation of the Gynostemma pentaphyllum butanol fraction (GPB)
G. pentaphyllum was cultivated by Thipsuta Co., Ltd., Thailand. The plant was authenticated (voucher specimen No. 15592) and preserved at the Herbarium of the Department of Biology, Chiang Mai University, Thailand. The leaves were shade dried and extracted with 95% EtOH at ambient temperature. The EtOH extract was then concentrated in vacuo, distilled water was added and the mixture washed with hexane and then chloroform. The aqueous fraction was then partitioned using n-butanol. The butanol phase was evaporated in vacuo to yield the GPB fraction as a dark brown residue (0.76% w/w of the dry starting material). In each experiment, the GPB was diluted with water to desired concentrations.
Male Sprague-Dawley rats weighing 150-200g each were purchased from the National Laboratory Animal Center, Salaya Mahidol University, Thailand. They were acclimatized for at least 7 days in an animal room where the temperature was maintained at 22[+ or -]3[degrees]C with a 12h light-dark cycle. The food was supplied by Pokphan Animal Feed Co., Ltd., Bangkok. The bedding was autoclaved. The rats had free access to food and water unless stated otherwise. All animals received humane care in compliance with the standards for the ethical use of animals issued by the National Research Council of Thailand 1999.
Indomethacin-induced gastric ulcers
GPB was administered orally to 48-h fasted rats, 60 min prior to induction of gastric ulcers by indomethacin suspended in 0.5% carboxymethylcellulose at a single i.p. dose of 30 mg/kg body wt. (Djahanguiri, 1969). After 5 h, the rats were sacrificed and examined for gastric ulcers.
HCl/EtOH-induced gastric ulcers
GPB was administered orally to 48-h fasted rats, 60 min prior to induction of gastric ulcers by 1.0 ml HCl/EtOH (60 ml EtOH + 1.7 ml HCl + 38.3 ml [H.sub.2]O) p.o. (Mizui and Doteuchi, 1988). The animals were sacrificed and examined for gastric ulcers 60 min later.
Restraint water-immersion stress-induced gastric ulcers
GPB was administered orally to 48-h fasted rats. Sixty minutes later, rats were restrained individually in stainless steel cages and immersed up to their xiphoid in a water bath maintained at 22[+ or -]2[degrees]C, according to the method of Takagi et al. (1963). After 5 h of this exposure, the rats were sacrificed and examined for gastric ulcers.
Evaluation of the gastric ulcers
After each rat was sacrificed, the stomach was removed, opened along the greater curvature and the glandular portion of the stomach was examined. The length in mm of each lesion was measured under a dissecting microscope and the sum of the length of all lesions was designated as the ulcer index.
GPB was administered orally to 48 h fasted rats. One hour later, pylorus ligation was performed as described by Shay et al. (1945). Briefly, rats were lightly anesthetized with ether. The abdomen was opened and the pylorus was ligated. The abdomen was closed by suturing. The animals were sacrificed 5 h later by an overdose of ether. The stomach was removed and its content was subjected to measurement of volume and pH and assayed for titratable acidity.
Determination of gastric wall mucus content
Gastric wall mucus was determined using the Alcian blue method (Corne et al., 1974). Briefly, GPB was administered orally to 48-h fasted rats, 60 min prior to induction of gastric ulcers by 1.0 ml HCl/EtOH (60 ml EtOH + 1.7 ml HCl + 38.3 ml [H.sub.2]O) p.o. (Mizui and Doteuchi, 1988). Sixty minutes later, the animals were sacrificed and the stomach was excised and opened along the lesser curvature, weighed and immersed in 0.1% w/v Alcian blue solution for 2 h. The excessive dye was then removed by two successive rinses in 0.25 M sucrose solution. Dye complexed with gastric wall mucus was extracted with 0.5 M Mg[Cl.sub.2] for 2 h. The blue extract was then shaken vigorously with an equal volume of diethyl ether and the resulting emulsion was centrifuged. The optical density of Alcian blue in the aqueous layer was read against a buffer blank at 580 nm using a spectrophotometer. The quantity of Alcian blue extract per gram wet stomach was then calculated from a standard curve.
Measurement of gastric hexosamine content
Hexosamine content in gastric tissue was assayed using the method of Glick (1967). Briefly, GPB was administered orally to 48-h fasted rats, 60 min prior to induction of gastric ulcers by 1.0 ml HCl/EtOH (60 ml EtOH + 1.7 ml HCl + 38.3 ml [H.sub.2]O) p.o (Mizui and Doteuchi, 1988). Sixty minutes later, the animals were sacrificed and the antral part of the stomach was hydrolyzed with 6N HCl overnight. The tissue was neutralized with 6N NaOH and incubated with acetylacetone at 100[degrees]C for 15 min. The mixture was then coupled with Ehrlich's reagent and allowed to stand at room temperature for 40 min. The optical density of the sample was measured spectrophotometrically at 530 nm using glucosamine as a standard.
Data were subjected to statistical analysis using ANOVA and statistical comparison was done using Duncan Multiple-Range Test. The value exceeding 95% confidence limits was considered to be significant.
Table 1 shows that GPB at a dose of 200 mg/kg body wt. inhibited ulcer formation induced by HCl/EtOH significantly, but failed to do so for the ulcers induced by indomethacin and water immersion. At a dose of 400 mg/kg body wt., GPB inhibited ulcers induced by indomethacin, HCl/EtOH and water immersion significantly (79, 94 and 78%, respectively), Remarkably, the inhibitory effect of GPB was obviously more profound in the HCl/EtOH model than in the other two models. In the pylorus-ligated rats, the mean gastric volume, gastric pH and acidity output were not affected significantly by GPB pretreatment (Table 2). Table 3 shows that the mean value of the gastric mucus content in HCl/EtOH-induced ulcerated rats was significantly lower than that of the control group. GPB at doses of 200 and 400 mg/kg body wt. significantly restored the mucus content back to a level comparable to that for the nonulcerated rats. Also in the assay for gastric hexosamine content, the mean gastric hexosamine content in control ulcerated rats was significantly less than that in the normal nonulcerated group, as shown in Table 4. Pretreatment with GPB at 200 and 400 mg/kg body wt. increased the hexosamine content significantly (39.5 and 51.7 [micro]g/100 mg wet stomach, respectively, compared to 21.7 [micro]g/100 mg wet stomach for the control HCl/EtOH ulcerated rats).
Results obtained in this study show the anti-gastric ulcer activity of GPB as evaluated in the most commonly utilized experimental models, including indomethacin, HCl/EtOH and restraint water-immersion stress-induced gastric lesions in rats (Robert et al., 1979; Murakami et al., 1985).
The pathogenesis of gastric ulcers is often depicted as an imbalance between mucosal integrity and aggressive factors. Factors that impair mucosal defense are HCl, gastrin, histamine, Helicobacter pylori, aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs), ethanol, caffeine and stress, while factors that promote mucosal integrity are gastric mucus and bicarbonate, gastric mucosal barrier, prostaglandins (PGs) and mucosal blood flow (Brunton, 1996; Friedman and Peterson, 1998).
According to the experimental models used in this study, NSAIDs like indomethacin induce ulcer formation by depleting cytoprotective PGs, e.g., PG[E.sub.2] and PG[I.sub.2], in the cyclooxygenase pathway of arachidonic acid metabolism (Satoh et al., 1981). PG[E.sub.2] and PG[I.sub.2] of gastric and duodenal mucosa are responsible for mucus production and maintaining cellular integrity of the gastric mucosa (Konturek et al., 1984). In the HCl/EtOH-induced gastric ulceration model, HCl causes severe damage to gastric mucosa (Yamahara et al., 1988), whereas ethanol produces necrotic lesions by direct necrotizing action which in turn reduces defensive factors like the secretion of bicarbonate and production of mucus (Marhuenda et al., 1993). The water-immersion stress-induced ulcers are mediated by increases in gastric acid secretion (Kitagawa et al., 1979) and decreases in mucosal microcirculation (Guth, 1972) and mucus content (Koo et al., 1986). Since the GPB could prevent ulceration in all three models and its anti-ulcer effect was much more pronounced in the ethanol model, it is not likely that inhibition of gastric secretion is the action of GPB. This is in accordance with the finding that the GPB could not significantly decrease gastric volume and increase gastric pH and acidity.
Gastric wall mucus, an obligatory component of which is hexosamines, is thought to play an important role as a defensive factor against gastrointestinal damage (Davenport, 1968). The determined gastric wall mucus was used as an indicator for gastric mucus secretion, while mucosal hexosamine content was used as an indicator for gastric wall mucus synthesis (Lukie and Forstner, 1972). In the present study, gastric wall mucus and hexosamine contents in HCl/EtOH ulcerated rats were markedly lowered as compared to those of the nonulcerated group. It was found that pretreatment with GPB increased both gastric mucus and hexosamine contents significantly in HCl/EtOH-ulcerated rats. This finding indicates that the GPB preserves both gastric mucus synthesis and secretion in the experimental rats.
In conclusion, this study provides evidence that the GPB obtained from G. pentaphyllum cultivated in Thailand possesses an anti-gastric ulcer effect which is related to a cytoprotective mechanism via preservation of gastric mucus synthesis and secretion.
Table 1. Effects of a Gynostemma pentaphyllum butanol fraction (GPB) on gastric ulcers in rats Group Gastric ulcer inducer Indomethacin HCl/EtOH Ulcer index (mm) I (%) Ulcer index (mm) Control 18.0 [+ or -] 4.4 58.2 [+ or -] 11.4 GPB 200 mg/kg body wt. 12.7 [+ or -] 2.4 29 3.7 [+ or -] 0.8* GPB 400 mg/kg body wt. 3.7 [+ or -] 1.0* 79 3.3 [+ or -] 0.7* Group Gastric ulcer inducer HCl/EtOH Stress I (%) Ulcer index (mm) I (%) Control 12.7 [+ or -] 1.9 GPB 200 mg/kg body wt. 94 6.8 [+ or -] 3.4 46 GPB 400 mg/kg body wt. 94 2.8 [+ or -] 0.6* 78 Note: data expressed as mean [+ or -] S.E.M. (n = 8); *significantly different from the control group (p<0.05); I(%) = inhibition of ulcer formation expressed as percentage. Table 2. Effects of a GPB fraction on gastric secretion in rats Group Gastric vol. (ml) Gastric pH Acidity (mEq/l) Control 7.6 [+ or -] 0.6 1.65 [+ or -] 0.06 202 [+ or -] 16 GPB 200 mg/kg 6.7 [+ or -] 2.4 2.04 [+ or -] 0.48 223 [+ or -] 22 body wt. GPB 400 mg/kg 8.1 [+ or -] 0.5 1.56 [+ or -] 0.03 184 [+ or -] 13 body wt. Note: data expressed as mean [+ or -] S.E.M. (n = 10); there were no significant differences. Table 3. Effects of a GPB fraction on gastric wall mucus content in rats Group Gastric wall mucus ([micro]g Alcian blue/g wet stomach) Control HCl/EtOH 804 [+ or -] 29 ulcerated rats GPB 200 mg/kg body wt. 1199 [+ or -] 43* GPB 400 mg/kg body wt. 1296 [+ or -] 56* Nonulcerated rats 1167 [+ or -] 16* Note: Data expressed as mean [+ or -] S.E.M. (n = 10); *significantly different from control ulcerated HCl/EtOH rats (p<0.05). Table 4. Effects of a GPB fraction GPB on gastric hexosamine content in rats Group Hexosamine content ([micro]g/ 100 mg wet stomach) Control HCl/EtOH 21.7 [+ or -] 1.6 ulcerated rats GPB 200 mg/kg body wt. 39.5 [+ or -] 1.9* GPB 400 mg/kg body wt. 51.7 [+ or -] 3.2* Nonulcerated rats 37.0 [+ or -] 2.2* Note: Data expressed as mean [+ or -] S.E.M. (n = 8); *significantly different from control HCl/EtOH ulcerated rats (p<0.05).
Received 3 January 2003; accepted 6 July 2003
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C. Rujjanawate*, D. Kanjanapothi, D. Amornlerdpison
Faculty of Medicine, Laboratory of Natural Products, Chulabhorn Research Institute, Chiang Mai University, Chiang Mai 5002, Thailand
E-mail address: firstname.lastname@example.org (C. Rujjanawate).
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|Title Annotation:||research findings|
|Author:||Rujjanawate, C.; Kanjanapothi, D.; Amornlerdpison, D.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Jul 1, 2004|
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